Proposed model for JNK regulation by <i>Drosophila</i> Mkk4 and Hemipterous/Mkk7 upon Eiger and Imd pathway activation.

<p>(A) Following Eiger binding to Wengen a signaling complex consisting of dTRAF2-dTAB2-dTAK1 is stabilized, which allows dTAK1 activation. Subsequently dTAK1 activates both Mkk4 and Hemipterous/Mkk7, which act non redundantly to activate Bsk/JNK. (B) Following Peptidoglycan recognition PGRP-LC recruits the scaffold protein IMD, which activates by a yet unknown mechanism the dTAB2-dTAK1 complex. dTAK1 subsequently activates directly the IKK complex and both Mkk4 and Hemipterous/Mkk7 which act non redundantly to activate Bsk/JNK.</p

Mkk4 function as a MAPKK between dTAK1 and JNK/Bsk.

<p>(A) RNAi against <i>Mkk4</i> and <i>hep</i> together significantly reduces dTAK1-induced AP-1-luciferase reporter activity. (B) In contrast to Hep and Hep^CA, Mkk4 and Mkk4^Asp do not induce AP-1-luciferase reporter activity on their own. (C) Mkk4 physically interacts with dTAK1 and Bsk. (D) Mkk4^Asp still interacts with dTAK1 but no longer binds to Bsk.</p

Mkk4 allelic series.

<p>Molecular lesions identified in <i>Mkk4</i>. Alleles are ordered according to their position in the protein.</p

Mkk4 triggers Eiger-mediated small eye phenotype.

<p>(A) Schematic representation of Mkk4. Alleles (black) and Ser/Thr phosphorylation sites (green) are indicated. (B)–(J) are in a <i>GMR-egr</i> (<i>GMR-Gal4</i>,<i>UAS-egr</i>) background. (B) <i>GMR-Gal4/+</i> control eye. (C) <i>GMR-egr/+</i> small eye. (D) <i>GMR-egr/+</i>; <i>Df(3L)Exel6149/+</i>. (E) <i>GMR-egr/+</i>; <i>Mkk4^G680/+</i>. Removing one copy of <i>Mkk4</i> suppresses the small eye phenotype. (F) <i>GMR-egr/+</i>; <i>Mkk4^G680/Df(3L)Exel6149</i>. Removing both copies of <i>Mkk4</i> does not improve the suppression. (G) <i>hep¹</i>; <i>GMR-egr/+</i>. Males hemizygous mutant for a hypomorphic <i>hep</i> allele display a strong suppression of the small eye. (H) <i>GMR-egr/tub-Mkk4</i>; <i>Mkk4^G680/+</i>. A <i>Mkk4</i> rescue transgene reverts the dominant suppression observed by loss of one copy of <i>Mkk4</i>. (I) <i>GMR-egr/UAS-Mkk4</i>. Co-expression of Mkk4 has dominant negative effect on Eiger signal transduction. (J) <i>GMR-egr/UAS-Mkk4^Asp</i>. The same effect is observed for Mkk4^Asp. (K)–(O) are in a <i>GMR-hep^CA</i> (<i>GMR-Gal4</i>,<i>UAS-hep^CA</i>) background. (K) <i>GMR-hep^CA/+</i> (25°C). (L) <i>GMR-hep^CA/UAS-Mkk4</i> (25°C). Co-expression of <i>Mkk4</i> also suppresses the small eye phenotype induced by Hep^CA. (M) <i>GMR-hep^CA/UAS-Mkk4^Asp</i> (25°C). Co-expression of <i>Mkk4^Asp</i> does not suppress the small eye phenotype induced by Hep^CA. (N) <i>GMR-hep^CA/+</i> (18°C). Weaker expression of <i>hep^CA</i> leads to a less severe small eye phenotype. (O) <i>GMR-hep^CA/+</i>; <i>Mkk4^G587/Mkk4^G673</i> (18°C). This phenotype is not suppressed, even when both copies of <i>Mkk4</i> are removed. (P) Aligment of the amino-acid sequence of Hep and Mkk4 catalytic region. The mutations introduced in Hep^CA, Mkk4^Asp and Mkk4^Mut are indicated in red.</p

Mkk4 activates the JNK branch of the Imd pathway, not the IKK/Relish branch.

<p>(A) RNAi against <i>Mkk4</i> and/or <i>hep</i> reduces phosphorylated JNK levels induced by commercial LPS in S2 cells. (B) Survival analysis of <i>w¹¹¹⁸</i> (closed diamond), <i>Mkk4^G673/Df(3R)Exel6149</i> (closed square)), <i>dTAK1¹</i> (cross) and <i>Relish^E20</i> (closed triangle) flies upon <i>Erwinia carotovora carotovora 15</i> (<i>Ecc15</i>) septic injury.</p

The Egr/dTAK1/Mkk4 cascade is dispensable to fight gram-positive cocci infection.

<p>(A) Survival analysis of <i>yw</i> (closed diamond), <i>w¹¹¹⁸</i> (closed square), <i>Mkk4^G673/Df(3R)Exel6149</i> (cross), <i>eiger³</i> (<i>egr</i>; closed triangle), <i>Spz^rm7</i> (closed circle) and <i>PGRP-SA^seml</i> (dash) flies upon <i>Staphylococcus aureus</i> (<i>S.aureus</i>) septic injury. (B) Quantitative RT-qPCR analysis of basal <i>egr</i> expression in <i>yw</i>, <i>Regg1</i>, <i>Regg1^1C</i>, <i>egr¹</i>, <i>egr³</i> and <i>egr⁶⁶</i> adults. ND: Not Detected. <i>RpL32</i> was used as the experimental expression standard. Relative DCt<i>^egr/</i>DCt<i>^RpL32</i> ratios of <i>yw</i> males were anchored in 1 to indicate fold-induction. Graphs represent the mean and S.D of relative ratios detected in 3 biological repetition of a pool of 15 males. (C–D) Survival analysis of <i>yw</i> (closed diamond), <i>eiger⁶⁶</i> (closed square), <i>eiger³</i> (cross), <i>egr¹</i> (closed triangle), <i>Regg1</i> (closed circle), <i>Regg1^1C</i> (dash) and <i>PGRP-SA^seml</i> (open circle) flies upon (C) <i>S.aureus</i> or (D) <i>Enterococcus faecalis</i> (<i>E.faecalis</i>) septic injury.</p

Microbiota-regulated genes.

<p>Microbiota-regulated genes are classified by Gene Ontology (GO):Biological function groups. Columns for each microbiota-regulated gene are designated as such: (1) Gene names from Flybase; (2) fold induction in microbiota associated animals vs GF; (3) information on gene function: lower hierarchy GO:Biological process or GO:Molecular function or insight on molecular function by mentioning an Interpro domain present in the protein; (4) Regulation by GATAe; (5) Gene deregulated upon sugar-only diet challenge; (6) Regulation by Sug; (7) Regulation by Myc; (8) Gene regulation in the midgut upon <i>Ecc15</i> infection (<i>Ecc15</i>) and (9) Relish dependence (Rel regulation) for gene expression in the midgut in basal condition (basal) or upon <i>Ecc15</i> infection (inf). For columns “GATAe” the data were collected from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094729#pone.0094729-Buchon3" target="_blank">[21]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094729#pone.0094729-Senger1" target="_blank">[27]</a>; columns “Sugar only” and “Sug” data are from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094729#pone.0094729-Zinke1" target="_blank">[29]</a>; column “Myc” data are from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094729#pone.0094729-Li1" target="_blank">[28]</a>; columns “<i>Ecc15</i>” and “Rel” data are from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094729#pone.0094729-Buchon4" target="_blank">[31]</a>.</p

<i>Drosophila</i> microbiota impacts midgut genes expression.

<p>(A) A schematic representation of the experimental set-up used for transcriptome analysis; GF: Germ-Free. (B) A volcano plot showing the absolute value of the computed statistics from the SAM analysis (d-stat) based on to the fold change in gene expression between the GF and poly-associated groups. The genes selected as differentially expressed by the SAM analysis are highlighted with a red circle around them. The selection was made using a value of Delta that corresponds to a FDR of 0.2. (C) Adult tissue enrichment classification of the 105 microbiota-regulated genes. (D) A bar graph representing the fold-enrichment of different microbiota-regulated transcripts in dissected midguts from GF vs poly-associated wild-type animals (the value of the relative ΔCt<i>^gene</i>/ΔCt<i>^rp49</i> ratio was calculated for every sample and relativized to the ratio in the GF condition which was anchored to 1 to indicate fold induction). Statistical significance of the result is represented (Student's t-test: ns≥0.05>*≥0.01>**≥0.001>***). Of note, <i>PGRP-SC1</i> mRNAs are detected to a 16 folds higher level than <i>PGRP-SC2</i> mRNA in young poly-associated animals (ΔCt<i>^PGRP-SC1</i>/ΔCt<i>^rp49</i> = 63 and ΔCt<i>^PGRP-SC2</i>/ΔCt<i>^rp49</i> = 4). (E) Relative percentage of Gene Ontology Terms: Biological processes in the list of microbiota-regulated gene (pie chart) and (F) Enrichment p-values (Benjamini-Hochberg corrected) for GO:Biological processes and GO:Molecular Functions in the list of microbiota-regulated genes.</p

IMD/Relish signaling controls microbiota mediated transcript enrichment in midguts.

<p>Bar graphs representing the fold enrichment of different microbiota-regulated transcripts in dissected midguts from GF vs poly-associated <i>Relish^E20</i> (A) or <i>Dredd^F64</i> (B) mutant animals (the values of the relative ΔCt<i>^gene</i>/ΔCt<i>^rp49</i> ratios were calculated for every sample and relativized to the ratio in the GF condition which was anchored to 1 to indicate fold induction). Statistical significance of the result is represented (Student's t-test: ns≥0.05>*≥0.01>**≥0.001>***). nd: not detected.</p